The spore-forming intestinal pathogen Clostridium difficile is the most prevalent cause of hospital-acquired infection in the developed world, whose financial cost approaches $5 billion per annum in the United States. C. difficile colonizes the large intestine of patients whose gut microbiota have been disrupted by antibiotic treatment. After colonization is established, C. difficile secretes protein toxins that disrupt cell-cell tigh junctions in the intestinal epithelium, causing inflammation, diarrhea, and potentially fatal pseudomembraneous colitis. C. difficile persists in the environment and infects new hosts in the form of metabolically dormant endospores, which are resistant to desiccation, oxidative stress, and most commercial disinfectants. C. difficile infection (CDI) is resistant to multiple antibiotic and has a high rate of recurrence. More effective therapies for CDI are sorely needed, but the organism is notoriously difficult to study and potential targets for therapeutic interventions are limited. My post-doctoral research focused on regulation of C. difficile colonization factors by th second messenger cyclic diguanylate. My future research will focus on the small signaling molecules guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp). Together (p)ppGpp regulate the stringent response, a conserved bacterial stress response that has never been studied in C. difficile. The C. difficile genome encodes functional (p)ppGpp metabolism genes, which are transcriptionally regulated by nutrient limitation and extracellular stress. The stringent response regulates toxin production, spore formation, and intrahost persistence in a number of pathogens, making it an attractive subject of investigation in C. difficile. I hypothesize that the (p)ppGpp-mediated stringent response regulates C. difficile survival and virulence. This proposal will (1) biochemically characterize (p)ppGpp metabolism in C. difficile, (2) define the environmental stresses that determine intracellular (p)ppGpp levels through transcriptional and post-translational regulation of (p)ppGpp metabolism, and (3) determine the roles of (p)ppGpp signaling in C. difficile virulence both by evaluating virulence traits in (p)ppGpp metabolism mutant strains in vitro and by assessing the colonization and lethality of these mutants in animal hosts. The proposed research will define the (p)ppGpp-mediated stringent response in C. difficile, identify virulence traits regulated by the stringent response, and identify potential targets for novel preventative and therapeutic strategies.